14 November, 2016 © Crown copyright

INTRODUCTION METHODS

RESULTS

• The three cell types displayed a similar morphology and the IncuCyte accurately

quantified confluence and cell number (fig 2).

• Cells reached mid-log phase after ~72 hours as determined by growth curve.

• IC50 values calculated using confluence (72 hours) or cell number (120 hours)

produced very similar IC50 values (fig 3, fig 4).

• A2780cis showed a ten-fold resistance to cisplatin.

• A2780ADR showed over ten-fold resistance to Adriamycin.

• These results are consistent with those published over 20 years ago generated

using different experimental techniques(1,3-6) (table 1).

DISCUSSION & CONCLUSION

• A2780cis and A2780ADR are an order of magnitude resistant to their respective therapeutic agents. These results

are consistent with the originally published data describing these cell lines(1+2).

• The IncuCyte ZOOM enabled the automation of imaging and quantification of confluence and nuclear count data.

• By comparing IC50 determination using cell number we have shown that cell confluence is a valid parameter to

determine IC50 in these cell lines.

• This study has verified that A2780 and its drug resistant counterparts, A2780cis and A2780ADR, have retained

resistance as reported over 20 years ago. The selective culture conditions used in the production of these cell lines

in ECACC has ensured the cells continue to be fit for purpose.

• This study has demonstrated the proof of principle of a label-free, fast and convenient way study in vitro drug

resistance.

ACKNOWLEDGEMENTS Special thanks go to Edward Burnett and Joseph Taylor of the

Scientific Development Group for on-going help and advice

relating to project work.

REFERENCES 1) Behrens, B. C. et al., 1987. 'Characterization of a cis-diamminedichloroplatinum(II)-resistant

human ovarian cancer cell line and its use in evaluation of platinum analogues', Cancer Res,

47: 414-8.

2) Behrens, B. C. et al., 1984. 'Resistance and cross-resistance of human ovarian cancer cell lines

to Adriamycin, melphalan, and irradiation', Proc. Am. Assoc. Cancer Res, 25: 336.

3) Masuda, H., R. et al., 1988. 'Increased DNA repair as a mechanism of acquired resistance to cis-

diamminedichloroplatinum (II) in human ovarian cancer cell lines', Cancer Res, 48: 5713-6.

4) Louie, K. G. et al., 1985. 'Radiation survival parameters of antineoplastic drug-sensitive and -

resistant human ovarian cancer cell lines and their modification by buthionine sulfoximine',

Cancer Res, 45: 2110-5.

5) Sasaki, H. et al., 1991. 'Human ovarian cancer cell lines resistant to cisplatin, doxorubicin, and L-

phenylalanine mustard are sensitive to delta 7-prostaglandin A1 and delta 12-prostaglandin

J2', Gynecol Oncol, 41: 36-40.

6) Hamilton, T. C. et al., 1984. 'Experimental model systems of ovarian cancer: applications to the

design and evaluation of new treatment approaches', Semin Oncol, 11: 285-98.

Andrew Bashford, Jim Cooper.

Scientific Development Group, Culture Collections, National Infection Service

Analysis of Drug Resistant Properties of A2780 Ovarian Cancer

Cell Lines Using Label-Free Automated Microscopy (IncuCyte ZOOM)

Drug Method

Time after

seeding

(hr)

IC50 values

A2780 (µM) A2780cis (µM) A2780ADR (µM)

Experimental IC50

Adriamycin

Cell number 120 0.007 0.016 0.070

Cell number

(DMSO normalised) 120 0.005 0.012 0.060

Confluence 72 0.004 0.023 0.071

Confluence

(DMSO normalised) 72 0.004 0.010 0.086

Range : 0.004 - 0.007 0.01 - 0.02 0.06 – 0.9

Published IC50 (Refs: 4, 5, 6)

Adriamycin Range: 0.01 - 0.1 - 1.6-710

Drug Method Time after

seeding (hr)

IC50 values

A2780 (µM) A2780cis (µM) A2780ADR (µM)

Experimental IC50

Cisplatin

Cell number 120 0.736 7.044 1.009

Confluence 72 0.438 7.812 0.997

Range : 0.4 - 0.7 7 - 8 1

Published IC50 (Refs: 1, 3, 5)

Cisplatin Range: 0.3 - 1.1 8-39 1.4

Adriamycin (doxorubicin) and cisplatin are frequently used cancer therapeutics which

interfere with DNA replication by different mechanisms. The A2780 series of ovarian

cancer cell lines are unique to The European Collection of Authenticated Cell Cultures

(ECACC) and its distributors. They have been effective in vitro tools for cancer

research for over 20 years(1,2).

A2780cis (ECACC 93112517) and A2780ADR (ECACC 93112520) (derivatives of A2780 (ECACC

93112519)) are resistant to cisplatin and Adriamycin respectively. They are useful in the

study of cancer biology and resistance to chemotherapies, enabling the search for new

treatments.

Drug resistance has been quantified in these cell lines using IC50 values; the dose of a

drug which causes a 50% inhibition in normal cell growth. The methods employed to

determine IC50 values often have different durations (4-21 days), they are labour

intensive and can yield inconsistent results(1-4).

Authenticated cell lines were provided from ECACC and grown

according to recommended instructions. Cells were seeded in 96-well

plates at densities of 3x104 cell/cm2 (A2780) and 1x104 cell/cm2

(A2780cis, A2780ADR) followed by addition of a drug dilution series.

• Cisplatin (P4394, Sigma) dissolved in PBS was diluted in media

(final well concentration: 500μM-0.001μM).

• Adriamycin (44583, Sigma) was dissolved in DMSO and diluted

in media, (final well concentration: 150μM-0.001μM). DMSO was

serially diluted in media to act as vehicle control.

Plates were incubated in the IncuCyte ZOOM (Essen BioScience).

Phase contrast images were obtained every two hours to provide data

for confluence determination. For cell number count, nuclear stain

Syto16 (S7578, Life Technologies) was added after 120 hours and

fluorescently imaged.

We used the two distinct parameters of confluence and cell count and

subsequent analysis using GraphPad Prism7 to determine IC50 values.

Adriamycin treatment was normalised using DMSO controls and no

difference was seen between IC50 values calculated using normalised or

raw values.

Table 1. IC50 values for A2780, A2780cis and A2780ADR (experimental and published data)

Fig 2) The IncuCyte

ZOOM is able to

accurately quantify

confluence and

nuclear count.

Representative images

from A2780 cells after

120 hr showing a)

phase data, b)

fluorescence of nuclear

stain Syto16. The

IncuCyte ZOOM trained

processing definition to

quantify c) percentage

confluence and d)

nuclear count as

number per mm2.

Scale bar: 70μm

Fig 1) The IncuCyte ZOOM

Fig 4) Cell number-

based IC50 values

show A2780cis and

A2780ADR have

higher drug

resistance than

parental A2780

cells. Error bars are

SEM generated from

6 (Adriamycin) or 8

(cisplatin) technical

replicates at each

drug concentration.

Fig 3) Confluence-

based IC50 values

show A2780cis and

A2780ADR have

higher drug

resistance than

parental A2780

cells. Error bars are

SEM generated from

6 (Adriamycin) or 8

(cisplatin) technical

replicates at each

drug concentration.

To independently characterise the cell lines and

investigate whether they have retained their drug

resistant properties, we developed a novel automated

approach using the IncuCyte ZOOM and a label-free

assessment of IC50 values. The study demonstrated a

proof of principle for the analysis of in vitro drug

resistance using cell lines.